Antimicrobial agents



ru wr- United States Eatent 2,908,606 ANTIMICROBIAL AGENTS Philip N.Gordon, Old Lyme, Conn., assiguor to Chas. Pfizer & Co., Inc., New York,N.Y., a corporation of Delaware N Drawing. Application July 25, 1956Serial No. 599,926

Claims. (Cl. 16733) This invention relates to certain novelantimicrobial agents prepared from streptomycin antibiotics. Moreparticularly, it is concerned with water-soluble complexes of copper andstreptomycin antibiotics, which are effective in combatting pathogenicor otherwise undesirable bacteria and fungi, especially plant pathogens.

Streptomycin is the name applied to a well-known antibiotic metabolismproduct of Streptomyces griseus, a microorganism generally found insoils. This antibiotic and its method of preparation are described inthe literature, for example in Waksman, Microbial Antagonisms andAntibiotic Substances, New York, The Commonwealth Fund, 1947, pages193-200. However, many forms of streptomycin are known, and it isintended to include within the scope of this invention streptomycin inall of its forms. Thus, the expression a streptomycin antibioticembraces not only streptomycin itself, but also its biologically activecompounds and derivatives, such as streptomycin sulfate, hydrochloride,nitrate, etc.; as well as the dihydroproduct, dihydrostreptomycin,dihydrostreptomycin sulfate, hydrochloride and the like.

In accordance with the present invention, highly useful complexes,possessing unusual biological activity, are formed by the interaction ofa streptomycin antibiotic and a water-soluble, inorganic cupric salt.The complexes of this invention not only retain the antibacterialactivity of the streptomycin antibiotic, but also, they exhibit a highdegree of antifungal activity as well. They possess unique propertieswhich could not be expected from the known properties of thestreptomycin antibiotics and copper salts from which they are prepared.The solubility of the resulting complexes in water renders themparticularly suitable for use in a wide variety of agricultural ice 1reaction proceeds readily at room temperature, although and industrialapplications where combined antibacterial and antifungal activity isdesired.

cupric salts may be employed in preparing the aforesaid complexes, suchas cupric sulfate, cupric chloride, cupric nitrate, cupric bromide, andthe like. These are advantageously present in the complexes in amolecular ratio of from about 1:2 to about 3:1. In other Words, it ispreferred to employ a proportion of one molecule of metallic salt to twomolecules of the streptomycin antibiotic based upon the streptomycincontent thereof, to a proportion of three of salt to one of theantibiotic. Stated dilferently, the complexes. preferably contain fromabout 0.5 to 3 mols of the copper salt per molof'strepto mycinantibiotic. Complexes in which the proportion of metallic salt to thestreptomycin antibiotic is less than about 0.5 do not have the highlydesirable properties of the preferred complexes. Particularly usefulcomplexes are prepared by employing a 1:1 molar ratio of salt toantibiotic.

The complexes of this invention are preferably prepared by dissolving anacid salt of a streptomycin antiit may be accelerated by moderatelyelevated temperatures, say up to 50 C. While adjustment of the pH of thereaction solution is not ordinarily necessary, the pH should bemaintained between from about 2 to 6. Upon the completion of thereaction, the resulting solution may be dried, as by application of avacuum or by freeze drying, to obtain the desired complex in dry form.The complexes may also be precipitated from methanolic solution by theaddition of a non-hydroxylic solvent, such as ether or hexane. Thecomplexes so obtained usually contain some water of hydration, but

thisv will depend upon the degree of drying to which the are subjected.The symbol X is employed herein to designate the number of molecules ofwater per molecule of complex, it being understood that X may vary from0 to 10. l

The preparation of the complexes of this invention is more particularlyillustrated by the following examples, which are not intended to imposeany limitation on the invention.

EXAMPLE I In like manner, streptomycin trihydrochloride (6.91, grams,i.e. 0.01 mol) was dissolved in methanol (300 ml.), followed by theaddition of cupric chloride dihydrate (1.71 grams, i.e. 0.01 mol) inmethanol (10 ml.). After about 30 minutes the solution was evaporated todryness to obtain 6.75 grams of a bright yellow complex having thefollowing formula:

In accordance with the procedure of Examplel, streptomycin sulfate (14.6grams, i.e. 0.01 mol) was dissolved in water (25 ml.), followed by theaddition of cupric chloride (3.41 grams, i.e. 0.02 mol). After 45minutes the color of the solution changed from blue-green toemerald-green. It was then freeze-dried to obtain 18.9 grams of ayellow-green complex of the following formu- EXAMPLE IV By followingthis same procedure the following complexes are readily prepared:

'[C;,, H O N (HCl 3 .CuCl .XH O C H O N (HCl .3 CuCl .XH O 21 39 12 '7)2 4) 3- 4'- 2 21 39 12 7) 2 4) s- C 4- 2 z i ss iz flH la 2- (N s)2' 221 aa 12 'i) 2 4) ss) 2- 2 The antibacterial activity of the foregoingcomplexes is illustrated by the test results set forth in the followingtable, which indicates the minimum inhibitory concentration ofrepresentative complexes, i.e. the amount required to inhibit the growthof various bacterial and 1 P .1. indicates partial inhibition.

It is apparent from the above tests that the complexes of this inventionretain the antibacterial activity of the streptomycin antibiotics ofwhich they are composed. However, they also exhibit a high order ofantifungal activity.

These combined activities render the complexes particularly unique forcombatting agricultural pathogens and for certain industrialapplications, as in water treatment, where the control of both fungi andbacteria is essential. The fact that such complexes can be used foragricultural purposes is quite unexpected, since it is well known thatwater-soluble forms of copper ordinarily cause phytotoxic effects, suchas burning, and for this reason, are generally to be avoided. In thiscase however, the factor of water solubility is a decided advantage,since it facilitates application of the complexes in the form of sprays,and contributes toward a systemic rather than merely surface action.

The efficacy of the complexes of this invention against phytopathogenswas determined in a series of foliage tests in which different types ofplants were treated with the complexes at various concentrations andsubjected to a number of plant diseases. In one of these tests, thestreptomycin complex was applied in a concentration of 2000, 400 and 80p.p.m. in the form of an aqueous spray which was permitted to dry. Theplant was bruised lightly on its leaves to form receptor sites forpossible infection. A broth culture of the phytopathogen was atomized onthe plants until the leaves were thoroughly wetted, thereby subjectingeach of thereceptor sites to infection. Seven days later the plants wereexamined for infection on the injured leaves to determine thc'degree ofinfection, as evidenced by the appearance of char acteristic spots atthe receptor sites. Untreated checks were also run. The percentagecontrol of early blight of tomato obtained with the streptomycinhydrochloridecopper chloride complex of Example II is set forth in thefollowing table:

Thus substantially complete control of the infection was obtained withas little as 80 p.p.m. of the complex. No phytotoxic effects were causedby the complex.

In like manner, the same streptomycin hydrochloridecopper chloridecomplex was tested to determine its effect in controlling halo blight ofbeans. The results of these tests are indicated below in Table III. A

Table 111 Percent Control of Halo Blight: Beans Complex 2,000 400 p.p.m.p.p.m. p.p.m.

In this case, complete control of halo blight was obtained with 400p.p.m. of the complex, without any adverse effects on the plants.

In like manner, the efficacy of the streptomycin sulfatecopper chloridecomplex of Example III and the streptomycin sulfate-copper sulfatecomplex of Example I was determined against the halo blight of beans.The results of these tests appear below in Table IV. I

Table IV Percent Control of Halo Blight: Beans Complex 400 200 100p.p.m. p.p.m. p.p.m.

(C21 a0012 1)z-(SOOs-ZCUCh 100 67 43 m uOuNflz.(SO4)3.2CI1SO4.- 98 87 6Check 0 0 0 Here, both complexes gave substantially complete control atconcentrations of 400 p.p.m. Again no toxic effects were observed.

The efficacy of the aforesaid complexes of Examples I Again a highdegree of control was obtained without toxic effects.

When tested against late blight of tomato, the foregoing compounds wereequally effective, as may be seen.

from the following table, wherein infection of the plants is rated asfollows: 0=none; 01=none to slight;

1= slight; 1-2= slight to moderate; 2=1noderate; A 2-3 :moderate tosevere; 3=severe.

Table VI Degree of Late Blight Infection Complex 400 p.p.m. so p.p.m;

(Czl se012N7)z.(5002011804 0 0-1 0 3 Thus substantially no infection wasobserved when the three complexes were applied at concentrations of 400p.p.m.; even at 80 p.p.m. one effected complete protection, whereas withthe others, infection was only slight. No phytotoxicity due to thecomplexes was observed.

Because of their high solubility in water, the complexes of thisinvention are readily incorporated in aqueous solutions to form sprayssuitable for application to plants, andthe like, to protect the sameagainst both bacterial and fungal infection. They may also beincorporated into Water systems where it is desired to control bacterialand fungal contamination. However, the application of these complexes isnot limited to spray forms, since emulsions, dusting powders,concentrates and the like may also be employed, wherein the activeingredient may range from about 1 to 95% by weight of composition on anactivity basis. These compositions may include inert carriers such aspyrophyllite, various clays, diatomaceous earth, etc. If desired, theymay also include Wetting agents and stabilizers, and in some instances,even insecticides.

The preparation of dry concentrates which may be diluted with waterimmediately before use, as in agricultural sprays, is particularlyadvantageous. Such concentrates may be composed of the aforesaidcomplexes or they may include suitable proportions of a water-soluble,inorganic cupric salt and a streptomycin antibiotic in uncomplexed form,which upon dissolution in water will form the desired complex in apredetermined concentration.

The amount and concentration of the complexes of this invention to beemployed against a particular plant disease, or to be employed forgeneral control of bacterial and fungal microorganisms, will, of course,vary considerably. In the agricultural field, the type of plant or tree,the disease involved, the time of year, Weather conditions, and thestage of development of the plant, are among the many factors which areto be considered in this respect. For most purposes, it is preferred toapply the streptomycin antibiotic-copper complex in concentrationsranging from about 80 to 500 ppm. to obtain the desired degree ofcontrol. It will be appreciated, however, that larger or smaller amountsmay be employed to meet the needs of individual situations.

What is claimed is:

1. A process for the preparation of a water-soluble complex of astreptomycin antibiotic and a Water-soluble, inorganic salt of copper,which comprises reacting a streptomycin antibiotic in a solvent selectedfrom the group consisting of methanol and water, with from about 0.5 to3 molecular proportions of water-soluble, inorganic cupric salt, whilemaintaining the reaction solution at a pH between about 2 and 6.

2. A water-soluble complex of a streptomycin antibiotic and from about0.5 to 3 molecular proportions of a water-soluble, inorganic cupricsalt, characterized by a high degree of antifungal activity.

3. A Water-soluble complex of streptomycin hydrochloride and about 1molecular proportion of cupric chloride, characterized by a high degreeof antifungal activity and having the approximate empirical formulaC31H3gO12N7.(HCl)3.Cl1C12.m2O wherein X is a number from 0 to 10.

4. A water-soluble complex of streptomycin sulfate and about 1 molecularproportion of cupric chloride, characterized by a high degree ofantifungal activity and having the approximate empirical formula (C H ON (S04) .CuCl .XH O

wherein X is a number from 0 to 10.

5. A water-soluble complex of streptomycin sulfate and about 1 molecularproportion of cupric sulfate, characterized by a high degree ofantifungal activity and having the approximate empirical formula(C21H39O12N7) (S0 .OuSO .XH O

wherein X is a number from 0 to 10.

6. A water soluble complex of streptomycin hydrochloride and about 1molecular proportion of cupric sulfate, characterized by a high degreeof antifungal activity and having the approximate empirical formulawherein X is a number from 0 to 10.

7. A water-soluble complex of streptomycin hydrochloride and about 1molecular proportion of cupric nitrate, characterized by a high degreeof antifungal activity and having the approximate empirical formulawherein X is a number from 0 to 10.

8. An aqueous spray for inhibiting the growth of bacteria and fungi,composed of an aqueous solution containing fromabout to 500 ppm. of awater-soluble complexof a streptomycin antibiotic and from about 0.5 to3 molecular proportions of a water-soluble, inorganic cupric salt.

9. A dry composition suitable for extemporaneous dissolution in water toform a streptomycin antibiotic-copper complex, characterized by a highdegree of antifungal activity, which comprises a streptomycin antibioticin physical admixture with from about 0.5 to 3 molecular proportions ofa Water-soluble, inorganic cupric salt.

10. A method for combatting industrial and agricultural bacteria andfungi which comprises applying to the habitat of such a microorganism, acomposition containing a water-soluble complex of a streptomycinantibiotic and from about 0.5 to 3 molecular proportions of awatersoluble, inorganic cupric salt.

Foye et al.: Journal of the American Pharmaceutical Association, vol.XLIV, May 1955, No. 5; pp. 261 to 263 relied on.

1. A PROCESS FOR THE PREPARATION OF A WATER-SOLUBLE COMPLEX OF ASTREPTOMYCIN ANTIBIOTIC AND A WATER-SOLUBLE, INORGANIC SALT OF COPPER,WHICH COMPRISES REACTING A STREPTOMYCIN ANTIBIOTIC IN A SOLVENT SELECTEDFROM THE GROUP CONSISTING OF METHANOL AND WATER, WITH FROM ABOUT 0.5 TO3 MOLECULAR PROPORTIONS OF WATER-SOLUBLE, INORGANIC CUPRIC SALT, WHILEMAINTAINING THE REACTION SOLUTION AT A PH BETWEEN ABOUT 2 AND 6.